Analyzing the Carotenoid Composition of Melilot (Melilotus officinalis (L.) Pall.) Extracts and the Effects of Isolated (All-E)-lutein-5,6-epoxide on Primary Sensory Neurons and Macrophages.

Melilotus officinalis is known to contain several types of secondary metabolites. In contrast, the carotenoid composition of this medicinal plant has not been investigated, although it may also contribute to the biological activities of the drug, such as anti-inflammatory effects. Therefore, this study focuses on the isolation and identification of carotenoids from Meliloti herba and on the effect of isolated (all-E)-lutein 5,6-epoxide on primary sensory neurons and macrophages involved in nociception, as well as neurogenic and non-neurogenic inflammatory processes. The composition of the plant extracts was analyzed by high performance liquid chromatography (HPLC). The main carotenoid was isolated by column liquid chromatography (CLC) and identified by MS and NMR. The effect of water-soluble lutein 5,6-epoxide-RAMEB (randomly methylated-ß-cyclodextrin) was investigated on Ca2+-influx in rat primary sensory neurons induced by the activation of the transient receptor potential ankyrin 1 receptor agonist to mustard-oil and on endotoxin-induced IL-1ß release from isolated mouse peritoneal macrophages. (all-E)-Lutein 5,6-epoxide significantly decreased the percent of responsive primary sensory neurons compared to the vehicle-treated stimulated control. Furthermore, endotoxin-evoked IL-1ß release from macrophages was significantly decreased by 100 µM lutein 5,6-epoxide compared to the vehicle-treated control. The water-soluble form of lutein 5,6-epoxide-RAMEB decreases the activation of primary sensory neurons and macrophages, which opens perspectives for its analgesic and anti-inflammatory applications.

Lutein inhibits breast cancer cell growth by suppressing antioxidant and cell survival signals and induces apoptosis.

Reduced risk of breast cancer upon intake of lutein-rich food supplements creates an interest to investigate the molecular mechanism underlying the growth inhibitory potential of lutein in MCF-7 and MDA-MB-231 cells. Lutein purified from Spinacia oleracea was identified by high-performance liquid chromatography and liquid chromatography-mass spectrometry. The cell viability was measured by water-soluble tetrazolium-1 assay. The intracellular reactive oxygen species level was examined by 2',7'-dichlorofluorescein assay. The protein expression of the markers of antioxidant defense, cell survival, and apoptosis was analyzed by western blot analysis. The induction of apoptosis by lutein was measured by 4',6-diamidino-2-phenylindole staining and caspase-3 activity assay. The purified lutein inhibited the viability of MCF-7 and MDA-MB-231 cells. The growth inhibitory effect of lutein was associated with suppressed protein expression of superoxide dismutase-2 and heme oxygenase-1, and its transcription factor nuclear factor erythroid 2-related factor-2. Lutein treatment subsequently blocked the expression of intracellular cell survival proteins, phosphorylated protein kinase B, phosphorylated extracellular-regulated kinase 1/2, and nuclear factor-kB. Suppression of antioxidant defense and cell survival markers by lutein was further linked to apoptosis induction with elevated caspase-3 activity and downregulated expression of Bcl-2 and poly-ADP ribose polymerase. Our results emphasize a significant role of lutein as an effective inhibitor of human breast cancer cell growth that activates cell death partly through the modulation of antioxidant defense response-linked cell survival signaling markers.

Improvement of carotenoid extraction from a recently isolated, robust microalga, Tetraselmis sp. CTP4 (chlorophyta).

In recent years, there has been increasing consumer interest in carotenoids, particularly of marine sustainable origin with applications in the food, cosmeceutical, nutritional supplement and pharmaceutical industries. For instance, microalgae belonging to the genus Tetraselmis are known for their biotechnologically relevant carotenoid profile. The recently isolated marine microalgal strain Tetraselmis sp. CTP4 is a fast-growing, robust industrial strain, which has successfully been produced in 100-m3 photobioreactors. However, there are no reports on total carotenoid contents from this strain belonging to T. striata/convolutae clade. Although there are several reports on extraction methods targeting chlorophytes, extraction depends on the strength of cell coverings, solvent polarity and the nature of the targeted carotenoids. Therefore, this article evaluates different extraction methods targeting Tetraselmis sp. CTP4, a strain known to contain a mechanically resistant theca. Here, we propose a factorial experimental design to compare extraction of total carotenoids from wet and freeze-dried microalgal biomass using four different solvents (acetone, ethanol, methanol or tetrahydrofuran) in combination with two types of mechanical cell disruption (glass beads or dispersion). The extraction efficiency of the methods was assessed by pigment contents and profiles present in the extracts. Extraction of wet biomass by means of glass bead-assisted cell disruption using tetrahydrofuran yielded the highest amounts of lutein and ß-carotene (622 ± 40 and 618 ± 32 µg g-1 DW, respectively). Although acetone was slightly less efficient than tetrahydrofuran, it is preferable due to its lower costs and toxicity.

Novel protocol optimized for microalgae lutein used as food additives.

Lutein available in the current market is derived from marigold petals. However, extensive studies showed that microalgae are rich in lutein content and potentially exploitable for its dietary and other industrial applications. In this study, microwave assisted binary phase solvent extraction method (MABS) was the novel protocol being developed and optimized to achieve maximum lutein recovery from microalgae Scenedesmus sp. biomass. Results showed that 60% potassium hydroxide solution with acetone in the ratio of 0.1 (ml/ml) was the ideal binary phase solvent composition. Empirical model developed using response surface methodology revealed highest lutein content can be recovered through MABS extraction method at 55 °C treatment temperature, 36 min in extraction time, 0.7 (mg/ml) for biomass to solvent ratio, 250 Watt microwave power and 250 rpm stirring speed. This optimized novel protocol had increased the amount of lutein recovered by 130% and shorten the overall extraction time by 3-folds.

Preparation and characterization of lutein ester-loaded oleogels developed by monostearin and sunflower oil.

The marigold (Tagetes erecta L.) flower is rich in lutein ester with many health-promoting activities. In this study, the effects of vegetable oil type and extracting the temperature on the extraction efficiency of lutein ester in the marigold flower were evaluated. Then, the structuring of the lutein ester-loaded sunflower oil with the addition of different amounts of monostearin and cooling temperatures (4 and 20°C) was investigated. The XRD analysis suggested that these oleogels were stabilized by the network formed by monostearin crystals in the sunflower oil. The textural properties (firmness, cohesiveness, and hardness) of oleogels were positively related to the monostearin dosage, but negatively related to the cooling temperature. According to the rheological results, the oleogels belonged to the pseudoplastic gel and their gelation temperature (Tg ) was only related to the concentration of monostearin. The light stability of lutein ester in the oleogels was also significantly improved in a monostearin dosage-dependent manner. PRACTICAL APPLICATIONS: The edible lutein ester-loaded oleogel for foods developed by structuring the sunflower oil with monostearin is introduced in this study. Its texture and rheological properties can be adjusted to cater to different requirements in the food industry by changing the monostearin dosage and cooling temperature. This study provides a reference for the development of other liposoluble nutraceuticals.

Optimization of Ultrasonic-Assisted Extraction and Purification of Zeaxanthin and Lutein in Corn Gluten Meal.

Zeaxanthin and lutein have a wide range of pharmacological applications. In this study, we conducted systematic experimental research to optimize antioxidant extraction based on detection, extraction, process amplification, and purification. An ultrasonic-assisted method was used to extract zeaxanthin and lutein with high efficiency from corn gluten meal. Firstly, the effects of solid-liquid ratio, extraction temperature, and ultrasonic extraction time on the extraction of zeaxanthin were investigated in single-factor experiments. The optimization extraction parameters of zeaxanthin and lutein with ethanol solvent were obtained using the response surface methodology (RSM) as follows: liquid-solid ratio of 7.9:1, extraction temperature of 56 °C, and extraction time of 45 min. The total content of zeaxanthin and lutein was 0.501%. The optimum extraction experimental parameters were verified by process amplification, and we confirmed that the parameters of the extraction process optimized using the RSM design are reliable and precise. Zeaxanthin and lutein from crude extract of corn gluten were separated and purified using silica gel column chromatography with the purity of zeaxanthin increasing from 0.28% to 31.5% (about 110 times) and lutein from 0.25% to 16.3% (about 65 times), which could be used for large-scale industrial production of carotenoids.

Antioxidant pectin enriched fractions obtained from discarded carrots (Daucus carota L.) by ultrasound-enzyme assisted extraction.

Carrot residues were upgraded as pectin-enriched fractions (PEFs) useful for functional food formulation due to co-extracted antioxidants (α- and ß-carotenes, lutein, α-tocopherol), and gelling effect. High power ultrasound (US)-enzyme assisted extraction was applied for efficiency and sustainability. Carrot powder (CP) in citrate-buffer (pH 5.20) was submitted to US-pretreatment (12.27 W/cm2: 20 kHz, 80% amplitude, 20 min) and a subsequent digestion (5 h-40 °C) without or with hemicellulase or cellulase. US-hemicellulase led to the highest PEF yield (27.1%), and extracted almost the whole pectin content of CP. US-pretreatment increased the extraction yield of all PEFs, but the existence of an additional positive effect of the following step depended on the enzyme used. PEFs contained 40-47% of UA with low DM (24-49.9%), and co-extracted antioxidants. US decreased the antioxidant contents, DM, and molecular weight, but allowed obtaining calcium crosslinked true gels, also with higher elastic modulus than non-US-extracted PEFs, being promising as food additives.

Supercritical Fluid Extraction of Lutein from Scenedesmus almeriensis.

Lutein has several benefits for human health, playing an important role in the prevention of age-related macular degeneration (AMD), cataracts, amelioration of the first stages of atherosclerosis, and some types of cancer. In this work, the Scenedesmus almeriensis microalga was used as a natural source for the supercritical fluid (SF) extraction of lutein. For this purpose, the optimization of the main parameters affecting the extraction, such as biomass pre-treatment, temperature, pressure, and carbon dioxide (CO2) flow rate, was performed. In the first stage, the effect of mechanical pre-treatment (diatomaceous earth (DE) and biomass mixing in the range 0.25-1 DE/biomass; grinding speed varying between 0 and 600 rpm, and pre-treatment time changing from 2.5 to 10 min), was evaluated on lutein extraction efficiency. In the second stage, the influence of SF-CO2 extraction parameters such as pressure (25-55 MPa), temperature (50 and 65 °C), and CO2 flow rate (7.24 and 14.48 g/min) on lutein recovery and purity was investigated. The results demonstrated that by increasing temperature, pressure, and CO2 flow rate lutein recovery and purity were improved. The maximum lutein recovery (~98%) with purity of ~34% was achieved operating at 65 °C and 55 MPa with a CO2 flow rate of 14.48 g/min. Therefore, optimum conditions could be useful in food industries for lutein supplementation in food products.

Marigold carotenoids: Much more than lutein esters.

Carotenoids constitute a large group of lipophilic pigments whose health-promoting benefits have been widely recognized. Hydroxy-containing carotenoids can be found in both free form or esterified with fatty acids in several plant matrices, but the native carotenoid profile is overall poorly explored due to the difficulty of analyzing carotenoid esters. One of the main natural sources of carotenoids is the marigold flower, which has been extensively used by the industry for the production of food colorants or supplements, both often manufactured with no saponification process. Although lutein esters are well established as the major compounds naturally found in marigold petals and their products, carotenoid esters other than the lutein ones have not been extensively examined. We carried out a comprehensive identification of carotenoids and carotenoid esters from marigold petals by LC-DAD-(APCI+)MS/MS. Whereas 18 carotenoids were identified in the saponified extract, 56 were identified when no saponification procedure was carried out: 6 free carotenoids, 20 monoesters and 30 diesters. This is the first time that esters of zeaxanthin, violaxanthin, auroxanthin, zeinoxanthin and ß-cryptoxanthin are identified in marigold. The structural information obtained through characteristic fragmentation patterns and diagnostic fragments in MS and MS/MS spectra (APCI+) sustained the differentiation between carotenoid esters with similar characteristics. Therefore, the separation of carotenoids by reversed-phase liquid chromatography using C30 columns in combination with DAD and APCI-MS/MS detection allowed high sensitivity and selectivity for carotenoid ester analysis.

Effect of refining on bioactive composition and oxidative stability of hazelnut oil.

In this study, the effect of refining process on the content of phytochemicals, antioxidant capacity and oxidative stability of hazelnut oil was investigated. The oil samples were taken at the consecutive steps of hazelnut refining process and analyzed for some compositional properties along with the antioxidant capacity and oxidative stability. The results have shown that, carotenoid content of the hazelnut oil was decreased during the refining process. The main carotenoids of hazelnut oil were found to be lutein and zeaxanthin and these compounds were lost completely during bleaching step of the refining. On the other hand, phenolic compounds and tocopherols were also partly removed from hazelnut oil to a degree. Loses in antioxidant compounds caused a clear decrease in antioxidant capacity measured in either the oils or polar extract of oils. Oxidative stability of the oil samples was measured by Rancimat method and it was found that neutralization caused an increase in oxidative stability compared to the crude oil. However, deodorization step caused a slight decrease in oxidative stability probably as a result of partial removal of tocopherols at this stage.

Simultaneous extraction and enrichment of polyphenol and lutein from marigold (Tagetes erecta L.) flower by an enzyme-assisted ethanol/ammonium sulfate system.

Enzyme-assisted aqueous two-phase extraction (EA-ATPE) using ethanol/ammonium sulfate system was investigated for total polyphenol (TP) and lutein from marigold flowers. The important factors were investigated by single factor experiment and response surface methodology combined with Box-Behnken design to optimize the operating parameters of EA-ATPE. The maximum yields of TP and lutein were 83.56 ± 0.69 mg g-1 and 5.59 ± 0.13 mg g-1, respectively. Compared with aqueous two-phase extraction and Soxhlet extraction (SE), the extraction yield of TP by EA-ATPE is 64.91% higher and the extract of EA-ATPE has better antioxidant activity. The pretreatment effect was also researched by scanning electron microscopy. Thus, EA-ATPE is an efficient method for extracting bioactive components from plants.

Ascorbic Acid Effectively Improved Lutein Extraction Yield from Australian Sweet Lupin Flour.

Lutein is a xanthophyll, a bioactive phytochemical that presents itself as colourful pigments in plants. Australian sweet lupin flour has been incorporated as a food ingredient in wheat bread and pasta to improve their sensory property and nutritional quality. However, the amount of lutein in lupin flour has not yet been determined. This is the first study to quantify naturally occurring lutein in Australian sweet lupin flour after the extraction efficiency was optimised. Several organic solvents (acetone, isopropyl alcohol, ethyl acetate and hexane), the use of an ultrasonic bath or a probe, the need for saponification and addition of ascorbic acid (served as antioxidant) were tested to compare the extraction yield. HPLC was employed to analyse lutein in flour. Lowest lutein (68 µg/100 g) was determined in the hexane extract. Samples extracted using an ultrasonic bath (126-132 µg/100 g) contained higher lutein than those extracted using a probe (84-109 µg/100 g). Saponified samples showed significantly less lutein (30-64 µg/100 g) than their respective non-saponified ones (122-134 µg/100 g). Without added ascorbic acid, lutein that was extracted into isopropyl alcohol was 143 µg/100 g and was higher than those released into acetone (92 µg/100 g). When ascorbic acid was added, measured lutein in the extracts of isopropyl alcohol (155 µg/100 g) and acetone (138 µg/100 g) increased by 8 and 33%, respectively. Our results suggested that the choice of extraction solvents and addition of ascorbic acid was crucial for quantitative analysis of lutein, so that the lutein content in lupin flour can be accurately reported.

Extraction of Astaxanthin and Lutein from Microalga Haematococcus pluvialis in the Red Phase Using CO2 Supercritical Fluid Extraction Technology with Ethanol as Co-Solvent.

Astaxanthin and lutein, antioxidants used in nutraceutics and cosmetics, can be extracted from several microalgal species. In this work, investigations on astaxanthin and lutein extraction from Haematococcus pluvialis (H. pluvialis) in the red phase were carried out by means of the supercritical fluid extraction (SFE) technique, in which CO2 supercritical fluid was used as the extracting solvent with ethanol as the co-solvent. The experimental activity was performed using a bench-scale reactor in semi-batch configuration with varying extraction times (20, 40, 60, and 80 min), temperatures (50, 65, and 80 °C) and pressures (100, 400, and 550 bar). Moreover, the performance of CO2 SFE with ethanol was compared to that without ethanol. The results show that the highest astaxanthin and lutein recoveries were found at 65 °C and 550 bar, with ~18.5 mg/g dry weight (~92%) astaxanthin and ~7.15 mg/g dry weight (~93%) lutein. The highest astaxanthin purity and the highest lutein purity were found at 80 °C and 400 bar, and at 65 °C and 550 bar, respectively.

Production, separation, and characterization of apo-luteinoids by LC-MS/MS.

It has been postulated that chemical or enzymatic catabolism of carotenoids could produce apo­carotenoids which have biological activity. Our objective was to generate and chemically characterize a series of apo­luteinoids (i.e. products resulting from the catabolism of lutein) which could putatively be found in vivo. Lutein was oxidized using potassium permanganate to produce a series of apo­luteinals/luteinone of subsequently shorter chain lengths, from apo­8'­luteinal to apo­11­luteinal. Sodium borohydride reduced this mixture into the corresponding alcohols (i.e. apo­luteinols). Similarly, Tollens' reagent was employed to oxidize the aldehyde series into carboxylic acids (i.e. apo­luteinoic acids). Mixtures of products were separated via HPLC and characterized in-line using photodiode array (PDA) and tandem mass spectrometry (MS-MS). A global HPLC-PDA-MS/MS method was developed to separate the products, and application of the methods to the symmetric xanthophyll zeaxanthin further confirmed the ε- and ß-ring species. These methods can be employed for the study of lutein oxidation products in plants, foods and biological samples.

Supercritical Carbon Dioxide Extraction of Astaxanthin, Lutein, and Fatty Acids from Haematococcus pluvialis Microalgae.

Haematococcus pluvialis microalgae in the red phase can produce significant amounts of astaxanthin, lutein, and fatty acids (FAs), which are valuable antioxidants in nutraceutics and cosmetics. Extraction of astaxanthin, lutein, and FAs from disrupted biomass of the H. pluvialis red phase using carbon dioxide (CO2) in supercritical fluid extraction (SFE) conditions was investigated using a bench-scale reactor in a semi-batch configuration. In particular, the effect of extraction time (20, 40, 60, 80, and 120 min), CO2 flow rate (3.62 and 14.48 g/min) temperature (50, 65, and 80 °C), and pressure (100, 400, and 550 bar.) was explored. The results show the maximum recovery of astaxanthin and lutein achieved were 98.6% and 52.3%, respectively, at 50 °C and 550 bars, while the maximum recovery of FAs attained was 93.2% at 65 °C and 550 bars.

Biological Active Ecuadorian Mango 'Tommy Atkins' Ingredients-An Opportunity to Reduce Agrowaste.

Mango is a commercially important tropical fruit. During its processing, peel and seed kernel are discarded as waste but they could be recovered as an excellent and cost-effective source of health-promoting ingredients. This study aimed to characterize some of them, including carotenoids like the provitamin A ß-carotene and lutein, with an interest beyond its role in eye health. Other health-promoting compounds like tocopherols and polyphenols were also evaluated, as well as the in vitro antioxidant capacity of mango by-products. Regarding isoprenoids, α-tocopherol was mainly found in the peels and carotenoids concentration was higher in the pulps. ß-carotene was the most abundant carotene in pulp and seed kernel, whereas peel was the only source of lutein, with violaxanthin the most abundant xanthophyll in the different mango organs tested. With regard to polyphenols, peels exhibited greater variability in its phenolic composition, being the total content up to 85 and 10 times higher than the pulp and seed kernels, respectively. On the other hand, peels also stood out for being a very rich source of mangiferin. Seed kernels and peels showed higher antioxidant capacity values than the pulps. These results contribute to the valorization of mango by-products as new natural ingredients for the pharma and food industries.

Protective effects of silk lutein extract from Bombyx mori cocoons on ß-Amyloid peptide-induced　apoptosis in PC12 cells.

Beta-amyloid (Aß) peptide, the hallmark of Alzheimer's disease (AD), invokes a cascade of oxidative damage to neurons and eventually leads to neuronal death. This study evaluated the protective effects of lutein extract from yellow cocoons of Bombyx mori, and its underlying mechanisms against was investigated to assess its protective effects and the underlying mechanisms against Aß25-35-induced neuronal cell death in cultured rat pheochromocytoma (PC12) cells. Aß25-35-induced neuronal toxicity is characterized by decrease in cell viability, increase in intracellular reactive oxygen species (ROS) production, activation of mitochondrial death pathway, and activation the phospholyration of mitogen-activated protein kinase (MAPKs) pathway. Pretreatment with silk lutein extract significantly attenuated Aß25-35-induced loss of cell viability, apoptosis, MAPKs pathway activation and ROS production. Taken together, our present study suggests that silk lutein extract protects PC12 cells from Aß25-35-induced neurotoxicity via the reduction of the ROS production, and subsequent attenuation of the mitochondrial death pathway and reduces the activation of the MAPK kinase pathways. This compound might beneficial as potential therapeutic agent to prevent or retard the development and progression of AD.

Process analysis and modeling of a single-step lutein extraction method for wet microalgae.

Lutein is a commercial carotenoid with potential health benefits. Microalgae are alternative sources for the lutein production in comparison to conventional approaches using marigold flowers. In this study, a process analysis of a single-step simultaneous extraction, saponification, and primary purification process for free lutein production from wet microalgae biomass was carried out. The feasibility of binary solvent mixtures for wet biomass extraction was successfully demonstrated, and the extraction kinetics of lutein from chloroplast in microalgae were first evaluated. The effects of types of organic solvent, solvent polarity, cell disruption method, and alkali and solvent usage on lutein yields were examined. A mathematical model based on Fick's second law of diffusion was applied to model the experimental data. The mass transfer coefficients were used to estimate the extraction rates. The extraction rate was found more significantly related with alkali ratio to solvent than to biomass. The best conditions for extraction efficiency were found to be pre-treatment with ultrasonication at 0.5 s working cycle per second, react 0.5 h in 0.27 L/g solvent to biomass ratio, and 1:3 ether/ethanol (v/v) with 1.25 g KOH/L. The entire process can be controlled within 1 h and yield over 8 mg/g lutein, which is more economical for scale-up.

Virgin Olive Oil Enriched with Lutein-Zeaxanthin from Spinacia oleracea.

The aim of this work consists of developing a technological process for elaborating a virgin olive oil enriched in lutein-zeaxanthin extracted from spinach, studying different parameters like temperature, time of extraction and different ratios (spinach-oil). It was observed that the amount of carotenoids extracted increased up to a maximum after 24 hours and decreased as the maceration time progressed up to 60 hours, resulting of biological degradation. It was also observed that as more spinach we added, as more lutein-zeaxanthin in the enriched virgin olive oil was obtained. The best results were obtained after 24 hours by using a 75:25 ratio at 30°C. Values of oxidative stability decreased drastically, as well as other parameters such as acidity; peroxides index and Ks were modified when the enriched virgin olive oil was subjected to 45°C for 24 hours of maceration. Thus, the present procedure constitutes a way to achieve an increase in the daily intake of beneficial compounds.

Adaption of an in vitro digestion method to screen carotenoid liberation and in vitro accessibility from differently processed spinach preparations.

Dark green leafy vegetables are primary food sources for lutein and ß-carotene, however these bioactives have low bioavailability. The effects of mechanical and thermal processing as well as fat addition and fat type on lutein and ß-carotene liberation and in vitro accessibility from spinach were investigated. Lutein liberation and in vitro accessibility were three-fold higher from spinach puree compared to whole leaves. Results for ß-carotene liberation were similar, whereas that of ß-carotene accessibility was only about two-fold. Steaming had no or a negative effect on carotenoid liberation. Fat addition increased ß-carotene liberation from raw and steamed puree, but reduced lutein liberation from steamed leaves and raw puree. Fat types affected ß-carotene differently. Butter addition led to a 2.5 fold increased liberation from raw spinach puree, while the effect of olive and peanut oil was significantly lower, but only minor effects were observed for lutein.